Neutrophils are the first responders to sites of infection and are fundamental to the anti-fungal response. This includes conditions of oropharyngeal candidiasis, where blood neutrophils extravasate into infected tissues and locomote to contact invading Candida. During this process, neutrophils are highly sensitive not only to the biochemical properties of the tissue microenvironment but the physical properties as well. The oral cavity is highly variant in the mechanical properties of its composite tissues, from the relatively soft and elastic tongue and soft palate to structures of relatively intermediate stiffness such as the esophagus to rigid structures such as the hard palate. The Reichner lab and others have shown that human neutrophils are highly sensitive to the mechanical features of their microenvironment including substrate stiffness. They have reported that human neutrophils move more slowly and with greater directionality when placed on stiff matrices as compared to soft matrices. Quantification and mapping of tractional forces show greater force generation on stiffer matrices with most of the force produced at the rear of the cell. A significant gap regarding these findings is that we have no information regarding how a pathogen, or its component PAMPs, affect the mechanosensitive response of neutrophils with regard to overall motility and the production of traction on matrices of varying stiffnesses. This is significant because it is the pathogen that induces neutrophil entry into an infected tissue to execute antimicrobial activity, and Candida infection is highly relevant in the oral cavity as a cause of OPC. The overarching hypothesis to be tested in this proposal is that the presence of candida affects the mechanosensitive properties of the human neutrophil. In Aim 1, we will use tunable elasticity hydrogels to determine how substrate stiffness regulates the neutrophil anti-Candida effector responses and, in turn, how the candida PAMP beta- glucan, affects the generation of traction force. Additionally, tissues are highly confined 3-dimensional spaces and, as such, are different than the 2D tissue culture substrates typically used for in vitro studies. Again, citing prior work from Reichner and colleagues, the physical property of confinement has a significant affect on neutrophil motility and traction. For example, whereas neutrophils are known to require integrins for adhesion and migration on 2D surfaces, integrins become dispensable for migration following entry into the highly confined 3D interstitial space. To study the sensitivity of neutrophils to physical confinement, we developed a double hydrogel compression device and have shown that confinement is the physical trigger for neutrophils to switch to integrin-independent migration and generation of traction. Therefore, although prior work has shown mechanosensitive effects on neutrophil integrin engagement in motility, nothing is known about whether confinement represents a mechanical property within tissues that regulates the anti-Candida effector functions (Aim 2).